Host-induced silencing of the CpCHI gene resulted in developmental abnormalities and mortality in maize stem borer (Chilo partellus).

RNAi-based insecticides for crop protection have witnessed rapid improvement over the years. However, their potential to efficiently control maize stem borer (Chilo partellus) pests has remained underexplored. In this study, double-stranded C. partellus chitinase (dsCHI) toxicity was investigated in C. partellus larvae. Furthermore, we developed transgenic maize lines expressing dsRNA targeted against C. partellus chitinase transcripts and performed detached leaf insect feeding bioassays. Our results revealed that C. partellus chitinase transcript expression was significantly downregulated by 57% and 82% in the larvae. Larvae exhibited various phenotypic distortion levels across developmental stages, and 53% mortality occurred in transgenic fed larvae compared to those fed on nontransgenic leaves. In conclusion, we have identified the C. partellus chitinase gene as a potential target for RNAi-mediated control and demonstrated that oral delivery via bacteria and plant-mediated delivery are viable means of achieving C. partellus RNAi-mediated control.

Enhancement of healthful novel sugar contents in genetically engineered sugarcane juice integrated with molecularly characterized ThSyGII (CEMB-SIG2).

Enhancement of sugar contents and yielding healthful sugar products from sugarcane demand high profile scientific strategies. Previous efforts to foster manipulation in metabolic pathways or triggering sugar production through combating abiotic stresses fail to yield high sugar recovery in Saccharum officinarum L. Novel sucrose isomers trehalulose (TH) and isomaltulose (IM) are naturally manufactured in microbial sources. In pursuance of novel scientific methodology, codon optimized sucrose isomerase gene, Trehalulose synthase gene II(CEMB-SIG2) cloned under dual combined stem specific constitutive promoters in pCAMBIA1301 expression vector integrated with Vacuole targeted signal peptide (VTS) to concentrate gene product into the vacuole. The resultant mRNA expression obtained by Real Time PCR validated extremely increased transgene expression in sugarcane culms than leaf tissues. Overall sugar estimation from transgenic sugarcane lines was executed through refractometer. HPLC based quantifications of Trehalulose (TH) alongside different internodes of transgenic sugarcane confirmed the enhancement of boosted sugar concentrations in mature sugarcane culms. Trehalulose synthase gene II receptive sugarcane lines indicated the unprecedented impressions of duly combined constitutive stem regulated promoters. Transgenic sugarcane lines produce highest sugar recovery percentages, 14.9% as compared to control lines (8.5%). The increased sugar recovery percentage in transgenic sugarcane validated the utmost performance and expression of ThSyGII gene .High Profile Liquid chromatography based sugar contents estimation of Trehalulose (TH) and Isomaltulose (IM) yielded unprecedented improvement in the whole sugar recovery percentage as compared to control lines.⁠.

Heterologous expression of cry1Ia12 insecticidal gene in cotton encodes resistance against pink bollworm, Pectinophora gossypiella (Lepidoptera: Gelechiidae); an alternate insecticidal gene for insect pest management.

BACKGROUND: Cotton is continuously exposed to sucking and chewing insect pest pressure since emergence to harvesting. Pink bollworm (Pectinophora gossypiella) has become major chewing insect pest to reduce the cotton yield and results in bad lint quality even in transgenic crops. The efficiency of insecticidal genes has been compromised due to extensive utilization of transgenic crops. METHODS AND RESULTS: The present study was conducted to evaluate the efficacy of an alternate cry1Ia12 insecticidal gene against pink bollworm (PBW) in cotton. Agrobacterium tumefaciens strain LBA4404 harboring pCAMBIA2300 expression vector containing cry1Ia12 gene under the control of 35S CaMV was used to transform a local cotton cultivar GS-01. The various molecular analyses revealed the transgene integration and expression in primary transformants. Among five selected transgenic plants, tcL-08 showed maximum (16.06-fold) mRNA expression of cry1Ia12 gene whereas tcL-03 showed minimum (2.33-fold) expression. Feeding bioassays of 2nd and 3rd instar pink bollworm (PBW) larvae on immature cotton bolls, flowers and cotton squares revealed up to 33.33% mortality on tcL-08 while lowest mortality (13.33%) was observed in tcL-03 and tcL-15. Furthermore, the average weight and size of survived larvae fed on transgenic plants was significantly lesser than the average weight of larvae survived on non-transgenic plants. CONCLUSIONS: The present study suggests the cry1Ia12 gene as an alternate insecticidal gene for the resistance management of cotton bollworms, especially PBW.

Silencing of multiple target genes via ingestion of dsRNA and PMRi affects development and survival in Helicoverpa armigera.

RNA interference (RNAi) triggered by exogenous double-stranded RNA (dsRNA) is a powerful tool to knockdown genetic targets crucial for the growth and development of agriculturally important insect pests. Helicoverpa armigera is a pest feeding on more than 30 economically important crops worldwide and a major threat. Resistance to insecticides and Bt toxins has been gradually increasing in the field. RNAi-mediated knockdown of H. armigera genes by producing dsRNAs homologous to genetic targets in bacteria and plants has a high potential for insect management to decrease agricultural loss. The acetylcholinesterase (AChE), ecdysone receptor (EcR) and v-ATPase-A (vAA) genes were selected as genetic targets. Fragments comprising a coding sequence of < 500 bp were cloned into the L4440 vector for dsRNA production in bacteria and in a TRV-VIGS vector in antisense orientation for transient expression of dsRNA in Solanum tuberosum leaves. After ingesting bacterial-expressed dsRNA, the mRNA levels of the target genes were significantly reduced, leading to mortality and abnormal development in larva of H. armigera. Furthermore, the S. tuberosum plants transformed with TRV-VIGS expressing AChE exhibited higher mortality > 68% than the control plants 17%, recorded ten days post-feeding and significant resistance in transgenic (transient) plants was observed. Moreover, larval lethality and molting defects were observed in larva fed on potato plants expressing dsRNA specific to EcR. Analysis of transcript levels by quantitative RT-PCR revealed that larval mortality was attributable to the knockdown of genetic targets by RNAi. The results demonstrated that down-regulation of H. armigera genes involved in ATP hydrolysis, transcriptional stimulation of development genes and neural conduction has aptitude as a bioinsecticide to control H. armigera population sizes and therefore decreases crop loss.

Silencing a Myzus persicae Macrophage Inhibitory Factor by Plant-Mediated RNAi Induces Enhanced Aphid Mortality Coupled with Boosted RNAi Efficacy in Transgenic Potato Lines.

Myzus persicae causes considerable losses to crops as a major pest. The damage is direct by feeding and also partly indirect because it vectors plant viruses. The currently available control strategies rely on unsafe and nonecofriendly chemical pesticide applications. Plant-mediated RNA interference (RNAi) has emerged as a powerful tool in crop protection from insect pests. Aphid salivary proteins are essential for phloem feeding and act as mediators of the complex interactions between aphids and their host plants. We documented the efficacy of dsRNA directed against macrophage inhibitory factor (MIF1) of M. persicae to induce aphid mortality and gene silencing through the generation of transgenic potato lines. A binary construct harbouring dsMIF1 driven by the CaMV35S promoter was introduced into the local potato variety 'AGB-white' by Agrobacterium-mediated transformation. PCR and Southern blotting validated the transgene presence and genomic integration in seven transgenic potato lines. An in vitro detached leaf assay revealed a significantly high aphid mortality of 65% in the transgenic potato line sDW-2, while the aphid mortality was 77% in the sDW-2 transgenic line during the in planta bioassay in comparison with 19% aphid mortality in the control nontransgenic potato line. A significantly high silencing effect was observed in the mRNA expression of MIF1, which was reduced to 21% in aphids fed on the transgenic potato line sDW-2. However, variable knockdown effects were found among six other transgenic potato lines, ranging from 30 to 62%. The study concluded that plant-mediated silencing of aphid RNA induces significant RNAi in M. persicae, along with enhanced aphid mortality.

Novel approaches to circumvent the devastating effects of pests on sugarcane.

Sugarcane (Saccharum officinarum L.) is a cash crop grown commercially for its higher amounts of sucrose, stored within the mature internodes of the stem. Numerous studies have been done for the resistance development against biotic and abiotic stresses to save the sucrose yields. Quality and yield of sugarcane production is always threatened by the damages of cane borers and weeds. In current study two problems were better addressed through the genetic modification of sugarcane for provision of resistance against insects and weedicide via the expression of two modified cane borer resistant CEMB-Cry1Ac (1.8 kb), CEMB-Cry2A (1.9 kb) and one glyphosate tolerant CEMB-GTGene (1.4 kb) genes, driven by maize Ubiquitin Promoter and nos terminator. Insect Bio-toxicity assays were carried out for the assessment of Cry proteins through mortality percent of shoot borer Chilo infuscatellus at 2nd instar larvae stage. During V0, V1 and V2 generations young leaves from the transgenic sugarcane plants were collected at plant age of 20, 40, 60, 80 days and fed to the Chilo infuscatellus larvae. Up to 100% mortality of Chilo infuscatellus from 80 days old transgenic plants of V2 generation indicated that these transgenic plants were highly resistant against shoot borer and the gene expression level is sufficient to provide complete resistance against target pests. Glyphosate spray assay was carried out for complete removal of weeds. In V1-generation, 70-76% transgenic sugarcane plants were found tolerant against glyphosate spray (3000 mL/ha) under field conditions. While in V2-generation, the replicates of five selected lines 4L/2, 5L/5, 6L/5, L8/4, and L9/6 were found 100% tolerant against 3000 mL/ha glyphosate spray. It is evident from current study that CEMB-GTGene, CEMB-Cry1Ac and CEMB-Cry2A genes expression in sugarcane variety CPF-246 showed an efficient resistance against cane borers (Chilo infuscatellus) and was also highly tolerant against glyphosate spray. The selected transgenic sugarcane lines showed sustainable resistance against cane borer and glyphosate spray can be further exploited at farmer's field level after fulfilling the biosafety requirements to boost the sugarcane production in the country.

Expression of ice recrystallization inhibition protein in transgenic potato lines associated with reduced electrolyte leakage and efficient recovery post freezing injury.

Potato (Solanum tuberosum L.) is considered to be frost-susceptible as short spells of frost can reduce the tuber yield and quality. Ice recrystallization inhibition (IRI) protein helps prevent growth of ice crystals in the cell apoplast during frost and help prevent damage associated with freezing stress. In this study, we investigated the in planta potential of Lolium perenne derived IRI3 transgene in improving the tolerance of transgenic potato lines for freezing stress. The codon optimized IRI3 transgene was introduced into potato cultivar Diamant through Agrobacterium mediated transformation. Three transgenic potato lines were successfully generated which were confirmed for transgene insertion and genomic integration by polymerase chain reaction and Southern blot. It was evident that the IRI3 transcript decreased in initial 24 h of cold stress treatment while the IRI3 mRNA expression up regulated in subsequent hours of cold treatment with maximum increase to 20 folds at 96 h post stress. A similar trend was also revealed in ion-leakage assay which showed that during cold stress, the transgenic potato lines depicted reduced ion leakage of 14-22% as compared to non-transgenic control plants. Further, the generated transgenic potato lines were tolerant to the frost spell in quarantine field conditions as compared to the non-transgenic potato lines. Additionally, the transgenic lines exhibited efficient recovery post frost injury in field conditions. The biochemical profiles of chlorophyll, proline and higher levels of antioxidant enzyme (superoxide dismutase, Catalase) activity and malondialdehyde levels showed that despite the phenotypic impact of low temperature, the transgenic potato lines quickly adjusted to maintain their cellular homeostasis post freezing stress by increasing the antioxidant defenses. This study suggests that up regulation of IRI3 transcript and regulatory network of cold stress response in transgenic potato lines improve frost tolerance and help stabilize yield in cultivated potato.

Advances in exogenous RNA delivery techniques for RNAi-mediated pest control.

Climate change imposes a great threat to world food security and encourages insect pest proliferation and spreading. Because of these challenges, identifying novel biotechnology pest management and its applications is inevitable. RNA interference (RNAi) is a gene regulatory process used for the maintenance and regulation of host defences against invading viruses. Nevertheless, it is widely used for the analysis of gene function. In recent years, the potential use of RNA interference (RNAi) as a tool for manipulating crop traits, as well as an alternative for crop protection, has undergone outstanding developments. In this review, we describe some genes involved in insect dsRNA uptake and discuss the reasons for varying RNAi response in insect pests, emphasizing the presence of nucleases and double-stranded RNA binding protein. We explore recent breakthroughs in innovative dsRNA delivery for efficient and effective knockdown in insect pests. Conclusively, topical delivery of dsRNA combined with a nanoparticle complex holds great potential for RNAi-mediated pest control.

Resistance to Chilo infuscatellus (Lepidoptera: Pyraloidea) in transgenic lines of sugarcane expressing Bacillus thuringiensis derived Vip3A protein.

Sustainable agriculture requires management of insect pests through resistance development. The biological potential of Cry toxins and Vip protein, derived from Bacillus species, is widely recognized in this context. The identification, evaluation of new insecticidal protein genes with different mode of action and entomotoxicity against sugarcane stem borer (Chilo infuscatellus) is important to overcome evolved insect resistance. In this study, we reported the generation of transgenic sugarcane lines expressing Vip3A toxin driven by polyubiquitin promoter for resistance against sugarcane stem borer. The V0 transgenic sugarcane plants were initially characterized by GUS histochemical staining, PCR and Southern blot assays that confirmed genetic transformation of twelve independent sugarcane lines. Variable transgene expression was found among transgenic sugarcane lines when revealed through Realtime quantitative PCR (RT-qPCR) with highest in S10 line while minimum was observed in V5 line. A similar expression pattern was observed in transgenic sugarcane lines for Vip3A protein concentration which ranged from 5.35 to 8.89 µg/mL. A direct correlation was observed between the Vip3A protein and Vip3A transgene expression in the transgenic sugarcane lines. In in-vitro insect bioassay on V1, Vip3A transgenic sugarcane lines exhibited high resistance to C. infuscatellus with upto 100% mortality compared to the control sugarcane line. Our findings suggest that a single copy insertion of Vip3A gene in transgenic sugarcane lines render them resistant to borer and these lines can be potentially used for generation of insect resistant transgenic sugarcane and could also be employed in gene pyramiding with Bt toxin to prolong resistance.

Report-Biochemical and molecular characterization of catalase enzyme in the saprobic fungus: Sordaria fimicola.

Fungi have been used in modern scientific research due to their high potential for different enzymes production based on genomic features. The great proportion of soil mycoflora represented by saprobic fungi plays an important role in decomposition, thus contribute to the global carbon cycle. Sordaria fimicola strains (n= 61) collected from different environments were evaluated for catalase enzyme activity at first stage. Among all 61 isolates of S. fimicola, five strains viz. S1, S2, N7, N6 and SF13 were found to be most efficient in catalase enzyme activity. The complete catalase gene including exons and introns was amplified and sequenced from the most efficient strains of S. fimicola and then submitted in the NCBI data base under accession numbers KM282183, KM282184, KM282186, KM282185 and KM282182 for strains S1, S2, N7, N6 and SF13 respectively. The significant differences in the genes sequences and theoretically translated proteins were observed for all five strains of S. fimicola. As regards catalase enzyme activity, S. fimicola strains were found comparable to the Aspergillus niger strains, therefore being a saprophytic fungus with short life cycle S. fimicola can become a fungus of choice to produce catalase enzyme at large scale.

Identification and validation of potential reference gene for effective dsRNA knockdown analysis in Chilo partellus.

Chilo partellus is an invasive polyphagous pest that has not been effectively managed with chemical pesticides. To select potential dsRNAs for use in an alternate control strategy, it is crucial to identify and evaluate stable reference genes for knockdown expression studies. This study evaluates the expression stability of seven candidate reference genes in C. partellus larvae fed on crude bacterially-expressed dsRNAs and purified dsRNAs at different time intervals, as well as the developmental stages and sexes. The expression stabilities of the reference genes were evaluated with different software programmes, such as BestKeeper, NormFinder, deltaCt, geNorm, and RefFinder. The overall results rank ELF as the most stably expressed reference gene when larvae were fed with crude bacteria-induced dsRNAs and purified dsRNA. However, Tubulin and HSP70 were more stable under different developmental stages and sexes. The expression levels of larvae that were fed crude bacteria-induced dsRNAs of Chitinase and Acetylcholinesterase were normalized with the four most stable reference genes (ELF, HSP70, V-ATPase and Tubulin) and the least stable reference gene (18S and HSP70) based on the geNorm algorithm. The least stable reference gene showed inconsistent knockdown expression, thereby confirming that the validation of a suitable reference gene is crucial to improve assay accuracy for dsRNA-targeted gene selection in C. partellus.

A Combinational Approach of Enhanced Methanol Production and Double Bt Genes for Broad Spectrum Insect Resistance in Transgenic Cotton.

The prevalence of insect resistance against Bt toxins has led to the idea of enhancing demethylation from cell wall pectin by pectin methylesterase enzyme for overproduction of methanol which is toxic to insects pests. The AtPME and AnPME fragments ligated into pCAMBIA1301 vector were confirmed through restriction digestion with EcoR1 and BamH1. Excision of 3363 bp fragment from 11,850 bp vector confirmed the ligation of both fragments into pCAMBIA1301 vector. Transformation of pectin methylesterase-producing genes, i.e., AtPME and AnPME from Arabidopsis thaliana and Aspergillus niger cloned in plant expression vector pCAMBIA1301 under 35S promoter into cotton variety CEMB-33 harboring two Bt genes Cry1Ac and Cry2A, respectively, was done by using shoot apex-cut Agrobacterium-mediated transformation method. The plantlets were screened on MS medium supplemented with hygromycin on initial basis. Amplification of 412 and 543 bp, respectively, through gene-specific primer has been obtained which confirmed the successful introduction of pCAMBIA AtPME and AnPME genes into cotton variety CEMB 33. Relative expression of AtPME and AnPME genes through real-time PCR determined the expression level of both gene ranges between 3- and 3.5-fold in different transgenic cotton lines along with quantity of methanol ranging from 0.8 to 0.9% of maximum while 0.5% to 0.6% of minimum but no expression was obtained in negative non-transgenic control cotton plant with least quantity of methanol, i.e., 0.1%. Almost 100% mortality was observed in insect bioassay for Helicoverpa armigera on detached leaves bioassay and 63% for Pink Bollworm (Pectinophora gossypiella) on growing transgenic cotton bolls as compared to positive control transgenic cotton with double Bt genes where mortality was found to be 82% for H. armigera and 50% for P. gossypiella while 0% in negative control non-transgenic plants.

Improved antifungal activity of barley derived chitinase I gene that overexpress a 32 kDa recombinant chitinase in Escherichia coli host

Abstract Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L.) cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935 bp full-length chitinase I gene. Based on the sequence of the amplified gene fragment, class I barley chitinase shares 93% amino acid sequence homology with class II wheat chitinase. Interestingly, barley class I chitinase and class II chitinase do not share sequence homology. Furthermore, the amplified fragment was expressed in Escherichia coli Rosetta strain under the control of T7 promoter in pET 30a vector. Recombinant chitinase protein of 35 kDa exhibited highest expression at 0.5 mM concentration of IPTG. Expressed recombinant protein of 35 kDa was purified to homogeneity with affinity chromatography. Following purification, a Western blot assay for recombinant chitinase protein measuring 35 kDa was developed with His-tag specific antibodies. The purified recombinant chitinase protein was demonstrated to inhibit significantly the important phytopathogenic fungi Alternaria solani, Fusarium spp, Rhizoctonia solani and Verticillium dahliae compared to the control at concentrations of 80 µg and 200 µg.

A virus-derived short hairpin RNA confers resistance against sugarcane mosaic virus in transgenic sugarcane.

RNA interference (RNAi) is commonly used to produce virus tolerant transgenic plants. The objective of the current study was to generate transgenic sugarcane plants expressing a short hairpin RNAs (shRNA) targeting the coat protein (CP) gene of sugarcane mosaic virus (SCMV). Based on multiple sequence alignment, including genomic sequences of four SCMV strains, a conserved region of ~ 456 bp coat protein (CP) gene was selected as target gene and amplified through polymerase chain reaction (PCR). Subsequently, siRNAs2 and siRNA4 were engineered as stable short hairpin (shRNA) transgenes of 110 bp with stem and loop sequences derived from microRNA (sof-MIR168a; an active regulatory miRNA in sugarcane). These transgenes were cloned in independent RNAi constructs under the control of the polyubiquitin promoter. The RNAi constructs were delivered into two sugarcane cultivars 'SPF-234 and NSG-311 in independent experiments using particle bombardment. Molecular identification through PCR and Southern blot revealed anti-SCMV positive transgenic lines. Upon mechanical inoculation of transgenic and non-transgenic sugarcane lines with SCMV, the degree of resistance was found variable among the two sugarcane cultivars. For sugarcane cultivar NSG-311, the mRNA expression of the CP-SCMV was reduced to 10% in shRNA2-transgenic lines and 80% in shRNA4-transgenic lines. In sugarcane cultivar SPF-234, the mRNA expression of the CP-SCMV was reduced to 20% in shRNA2-transgenic lines and 90% in shRNA4 transgenic lines, revealing that transgenic plants expressing shRNA4 were almost immune to SCMV infection.

Improved antifungal activity of barley derived chitinase I gene that overexpress a 32kDa recombinant chitinase in Escherichia coli host.

Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L.) cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935bp full-length chitinase I gene. Based on the sequence of the amplified gene fragment, class I barley chitinase shares 93% amino acid sequence homology with class II wheat chitinase. Interestingly, barley class I chitinase and class II chitinase do not share sequence homology. Furthermore, the amplified fragment was expressed in Escherichia coli Rosetta strain under the control of T7 promoter in pET 30a vector. Recombinant chitinase protein of 35kDa exhibited highest expression at 0.5mM concentration of IPTG. Expressed recombinant protein of 35kDa was purified to homogeneity with affinity chromatography. Following purification, a Western blot assay for recombinant chitinase protein measuring 35kDa was developed with His-tag specific antibodies. The purified recombinant chitinase protein was demonstrated to inhibit significantly the important phytopathogenic fungi Alternaria solani, Fusarium spp, Rhizoctonia solani and Verticillium dahliae compared to the control at concentrations of 80µg and 200µg.

Antifungal activity of chitinase II against Colletotrichum falcatum Went. causing red rot disease in transgenic sugarcane.

We evaluated transgenic lines of sugarcane modified with the barley chitinase class-II gene to create resistance against the red rot causative agent Colletotrichum falcatum Went. Local sugarcane cultivar SP93 was transformed with a 690-bp coding sequence of the chitinase-II gene under the influence of a polyubiquitin promoter. Transgenic sugarcane lines (T 0) overexpressing the chitinase gene were obtained through a particle bombardment method with 13.3% transformation efficiency. Four transgenic sugarcane lines, SCT-03, SCT-05, SCT-15, and SCT-20, were tested for resistance against red rot by in vitro antifungal assays. Crude protein extracts from transgenic sugarcane plants SCT-03, SCT-05, SCT-15, and SCT-20 inhibited the mycelial growth of C. falcatum by 49%, 40%, 56%, and 52%, respectively, in a quantitative in vitro assay. Our findings revealed that two transgenic lines, SCT-15 and SCT-20, exhibited the highest endochitinase activity of 0.72 and 0.58 U/mL, respectively. Furthermore, transgenic lines SCT-15 and SCT-20 exhibited strong resistance against inoculated C. falcatum in an in vitro bioassay, as they remained healthy and green in comparison with the control sugarcane plants, which turned yellow and eventually died 3 weeks after infection. The mRNA expression of the transgene in the C. falcatum-inoculated transgenic sugarcane lines increased gradually compared to the control plant. The mRNA expression was the highest at 72 h in both transgenic lines and remained almost stable in the subsequent hours.

Role of Molecular Biology in Cancer Treatment: A Review Article.

BACKGROUND: Cancer is a genetic disease and mainly arises due to a number of reasons include activation of onco-genes, malfunction of tumor suppressor genes or mutagenesis due to external factors. METHODS: This article was written from the data collected from PubMed, Nature, Science Direct, Springer and Elsevier groups of journals. RESULTS: Oncogenes are deregulated form of normal proto-oncogenes required for cell division, differentiation and regulation. The conversion of proto-oncogene to oncogene is caused due to translocation, rearrangement of chromosomes or mutation in gene due to addition, deletion, duplication or viral infection. These oncogenes are targeted by drugs or RNAi system to prevent proliferation of cancerous cells. There have been developed different techniques of molecular biology used to diagnose and treat cancer, including retroviral therapy, silencing of oncogenes and mutations in tumor suppressor genes. CONCLUSION: Among all the techniques used, RNAi, zinc finger nucleases and CRISPR hold a brighter future towards creating a Cancer Free World.

Engineered Disease Resistance in Cotton Using RNA-Interference to Knock down Cotton leaf curl Kokhran virus-Burewala and Cotton leaf curl Multan betasatellite Expression.

Cotton leaf curl virus disease (CLCuD) is caused by a suite of whitefly-transmitted begomovirus species and strains, resulting in extensive losses annually in India and Pakistan. RNA-interference (RNAi) is a proven technology used for knockdown of gene expression in higher organisms and viruses. In this study, a small interfering RNA (siRNA) construct was designed to target the AC1 gene of Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu) and the ßC1 gene and satellite conserved region of the Cotton leaf curl Multan betasatellite (CLCuMB). The AC1 gene and CLCuMB coding and non-coding regions function in replication initiation and suppression of the plant host defense pathway, respectively. The construct, Vß, was transformed into cotton plants using the Agrobacterium-mediated embryo shoot apex cut method. Results from fluorescence in situ hybridization and karyotyping assays indicated that six of the 11 T1 plants harbored a single copy of the Vß transgene. Transgenic cotton plants and non-transgenic (susceptible) test plants included as the positive control were challenge-inoculated using the viruliferous whitefly vector to transmit the CLCuKoV-Bu/CLCuMB complex. Among the test plants, plant Vß-6 was asymptomatic, had the lowest amount of detectable virus, and harbored a single copy of the transgene on chromosome six. Absence of characteristic leaf curl symptom development in transgenic Vß-6 cotton plants, and significantly reduced begomoviral-betasatellite accumulation based on real-time polymerase chain reaction, indicated the successful knockdown of CLCuKoV-Bu and CLCuMB expression, resulting in leaf curl resistant plants.

HIV Diagnosis and Treatment through Advanced Technologies.

Human immunodeficiency virus (HIV) is the chief contributor to global burden of disease. In 2010, HIV was the fifth leading cause of disability-adjusted life years in people of all ages and leading cause for people aged 30-44 years. It is classified as a member of the family Retroviridae and genus Lentivirus based on the biological, morphological, and genetic properties. It infects different cells of the immune system, such as CD4+ T cells (T-helper cells), dendritic cells, and macrophages. HIV has two subtypes: HIV-1 and HIV-2. Among these strains, HIV-1 is the most virulent and pathogenic. Advanced diagnostic methods are exploring new ways of treatment and contributing in the reduction of HIV cases. The diagnostic techniques like PCR, rapid test, EIA, p24 antigen, and western blot have markedly upgraded the diagnosis of HIV. Antiretroviral therapy and vaccines are promising candidates in providing therapeutic and preventive regimes, respectively. Invention of CRISPR/Cas9 is a breakthrough in the field of HIV disease management.

Amplicon-Based RNA Interference Targeting V2 Gene of Cotton Leaf Curl Kokhran Virus-Burewala Strain Can Provide Resistance in Transgenic Cotton Plants.

The conserved coat or V2 gene of begomoviruses is responsible for viral movement in the plant cells. RNAi technology was used to silence V2 gene for resistance against these viruses in transgenic plants. The transformation of the RNAi-based gene construct targeting V2 gene of CLCuKoV-Bur, cloned under 35S promoter, was done in two elite cotton varieties MNH-786 and VH-289 using shoot apex cut method of gene transformation. The transformation efficiency was found to be 3.75 and 2.88 % in MNH-786 and VH-289, respectively. Confirmation of successful transformation was done through PCR in T 0, T 1, and T 2 generations using gene-specific primers. Transgenic cotton plants were categorized on the basis of the virus disease index in T 1 generation. Copy number and transgene location were observed using FISH and karyotyping in T 2 generation which confirmed random integration of V2 RNAi amplicon at chromosome 6 and 16. Real-time quantitative PCR analyses of promising transgenic lines showed low virus titer compared to wild-type control plants upon challenging them with viruliferous whiteflies in a contained environment. From the results, it was concluded that amplicon V2 RNAi construct was able to limit virus replication and can be used to control CLCuV in the field.